Exam 2 Super Review Flashcards by Laura Pitt

i. Media: Sim tube (inoculate in a straight light)
ii. Reagent: None added
iii. Results
1. Tube is cloudy if mobile
2. Black means positive for H2S (the microbe is anaerobic)

Motility/H2S Test

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a. Media: Starch agar (Tan)
b. Reagent Added: Iodine
c. Results for Positive Test: Clear halo

Starch Hydrolysis

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Starch is hydrolyzed in to maltose, glucose and dextrins by

Amylase

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a. Media: Milk Agar (white)
b. Reagent Added: none
c. Results for Positive Test: Clear halo

Casein hydrolysis

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Fat, hydrolyzed into fatty acids and glycerol

lipase

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a. Media: DNA agar (aqua green compared to Spirit)
b. Reagent Added: none
c. Results for Positive Test: Clear Halo

DNA hydrolysis

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Hydrolyzed nucleotides

DNAse

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a. Media: Tryptophan Broth (in to test tube)
b. Reagent Added: Kovacs’ Solution (testing for presence of indole)
c. Results for Positive Test: Red/pink layer on top of the test tube

Tryptophan hydrolysis

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An amino acid, hydrolyzed into indole, pyruvic acid and ammonia by

tryptophanase

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a. Media: Urea Broth
b. Reagent Added: None
c. Results for Positive Test: Turns from yellow to pink (response to high pH)

Urea hydrolysis

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Hydrolyzed into ammonia and carbon dioxide by

urease

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a. Media: Bile Esculin Slant
b. Reagent Added: None
c. Results for Positive Test: Turns black

Esculin hydrollysis

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Hydrolyzed into glucose, esculatin and ferric citrate (dark brown salt) by

esculinase

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Media: Durham Fermentation Tube
Reagent Added:
Results: Yellow = acid; +/- for gas (see inverted tube)

Sugar Fermentation

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Media: MRVP Broth
Reagent Added: 5 drops of Methyl Red
Results: Red if pH 5

Mixed Acid Fermentation

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Media: Citrate Slant
Reagent Added:
Results: From green to intense blue

Citrate Utilization

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a. Citrate used during the kreb’s cycle
b. Suggests that they engage in some form of cellular respiration if they can ingest citrate
2. Media: Citrate Slant

Citrate Utilization

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Media: Nutrient Agar Tube
Reagent Added: Drop/o Oxidase on swab
Results: from Red=> Blue/Black

Oxidase Test

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a. Bacteria’s ability to has electron transport system

b. Suggests bacteria be engage in aerobic cellular respiration

Oxidase Test

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Media: nitrate broth
Reagent Added: 5 drops of nitrate A+B
Results: Red = presence of nitrite

Nitrate Reduction

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a. Nitrate provides oxygen for cells that engage in anaerobic cellular respiration
b. Red means anaerobic cellular respiration, since nitrate  nitrite in cellular respiration

Nitrate Reduction

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Media: MRVP
Reagent Added: VPA 15 drops, VPB 5 drops to 1 mL broth “Barritt’s reagent” tests for acetoin, precursor to alcohol fermentation product
Results: Red after 10 min

Alcohol Fermentation

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Media: Slant Agar
Reagent Added: Catalase (Hydrogen Peroxide)
Results: Bubbles or not

Catalase

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a. Bubbling indicates oxygen production, confirms the presence of catalase
b. Catalase is needed to survive in the presence of oxygen

Catalase

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1. plasmid 2. codes for beta-lactamase 3. and beta-galactosidase

PGal

a. test if transformed, put on agar with ampicillin | b. will enter media, inactivate ampicillin, normal sensitive can grow

beta lactamase

a. put e coli on agar enrihched with sugars galactosides | b. exoenzyme will enter media, hydrolyze sugars, turn blue

beta galactosidase

xii. =the natural process by which some bacteria can increase their genetic variability

Transformation

1. Being able to acquire naked DNA from the environment around

Competent

i. Put bacteria on the plate ii. Incubate on ice for 10 minutes iii. Remove and incubate in 42 degrees hot water bath for 90 seconds iv. Return tubes to ice water bath for 1 minute v. Add recovery broth to the tubes vi. Incubate in 37 degree water bath for 30 minutes vii. Inoculate controls. viii. Make E. Coli competent try to transform competent cells from white, ampicillin sensitive bacterium to a blue ampicillin resistant bacterium. ix. Mix E coli with plasmid pGal

Transformation

1. Streptococcus pyogenes 2. Staphylococcus aureus 3. Clear halo forms 4. All the RBC’s and hemoglobin have been destroyed

Beta hemolysis | Alcohol on Bacterial Growth

1. Halo generated, not clear 2. Billiverdin a. Prouct of hemoglobin hydrolysis

Alpha Hemolysis

Do not generate a halo

Gamma hydrolysis

i. Chemically defined media | ii. We know every component and how much of that component is in the media

Synthetic

i. Infusions and extracts ii. We use this in class a lot iii. Secretions of ground up animal/plant stuff iv. We don’t know the exact amount of each component beause we just kind of throw stuff in there. v. Most commonly used media

Non Synthetic

a. Liquid medium containing beef extract and peptone

Nutrient Broth

a. Solid media containing beef extract, peptone, and agar

Nutrient Agar

i. Media that prevents the growth of one type of bacteria without inhibiting the growth of another type

Selective

i. Media Select for G- | ii. Inhibit G+

EMB and Hektoen

i. Selects for halophiles

SM 110 | MSA Mannitol Salt agar

i. The way the organism grows on or its effect on a media helps tell the bacteria apart

Differential

i. Media Changes colors of bacteria

ChromAgar

a. EMB b. Blood agar c. ChromAgar d. MSA e. Columbia CNA

Examples of Differential Media

i. Additivevs are included to promote growth of fastidious bacteria 1. Ex: a. TSA b. Blood agar

Enriched media

good for propagating large numbers of organisms as well as for testing

Liquid media

shows surface growth patterns | i. Convenient for “pure culturing” organisms

Solid media

1. Just the protein alone 2. Has Apoenzyme core 3. Cofactors 4. Coenzymes

Simple Enzyme Structure

a. The protein portion | b. Contains the active site

Apoenzyme core

a. Non protein portions chemical compound that is bound tightly to an enzyme and is required for catalysis. i. Metallic 1. Fe 2. Cu 3. Mg

Cofactors

a. Organic materials, non protein molecule that carries chemical grous between enzymes b. vitamins

Coenzymes

i. They increase the rate of chemical reactions by lower the energy of activation.

Enzymes

ii. Only catalyze energy releasing reactions (net reaction must yield energy)

Enzymes

i. = a molecule with a shape complementary to the enzyme’s active site that, when it interacts with the enzyme, is changed by the enzyme

Substrate

ii. It binds to an active site.

Substrate

1. Enzymes that do their job intracellularly (inside the cell)

Endoenzymes

1. Enzymes that are secreted and do their jobs outside of the cell (extracellularly)

Exoenzymes

1. Enzymes that are produced in response to the presence of a particular substrate

Induced Enzymes

2. Only produced when the need is there; Turned on with changes in substrate concentration

Induced Enzymes

1. Need is always there | 2. Enzymes that are always produced

Constitutive Enzyme

1. Enzymes that are not produced when the product of the enzyme pathway is present

Repressible

2. Turned off in response to the substrate concentration

Repressible

vi. Their names are typically given based on their substrate and/or the reaction they complete

Enzymes

i. = Condensation Reactions ii. When larger molecules are synthesized from building blocks by removing water 1. Ex: proteins from amino acids

Dehydration synthesis

i. When large molecules are split apart (digested) by adding water

Hydrolysis

i. When a molecule loses electrons/hydrogens (OIL RIG)

Oxidation

i. When a molecule gains electrons | 1. Ex: during photosynthesis. CO2 is reduced to glucose C6H12O6

Reduction

i. When a phosphate is added to a molecule | 1. Ex: Photosynthesis: When chlorophyll used in conversion of ADP --> ATP

Phosphorylation

i. Occurs when the final product, of an enzyme pathway, blocks further enzyme activity ii. The product reacts with an allosteric site iii. The shape of the enzyme’s active site is changed; and product production ceases.

Feedback Inhibition

i. An inhibitor molecule resembles the enzyme’s normal substrate and competes with the substrate for the active site.

Competitive Inhibition

i. Resemble and compete with PABA for a bacterial enzyme’s active site ii. Is normally converted to the vitamin folic acid, by microbial enzymes.

Sulfa Drugs

adenine and guanine

Purines | Two rings

Cytosine and Thymine

Pyrimidines | One ring

i. 1 of the 4 nitrogenous bases ii. Deoxyribose: a 5 carbon sugar iii. A single phosphate group

Components of nucleotides

i. Sides of the DNA ladder consist of deoxyribose molecules alternating with phosphate molecules ii. Rungs (steps) of the ladder are made by nitrogenous bonds 1. A binds to a T/U (D/R NA) 2. C binds to a G

Arrangement of nucleotides

three base sequence that codes for an amino acid or a control system

codon

a sequence of codons between a start and stop codon that codes for a protein/RNA

gene

a cluster of genes (primarily in prokarya) that operate as a unit, e.g., genes for enzymes of the same biosynthetic pathway.

operon

one DNA molecule

chromosome

the sum total of all the chromosomal DNA in the cell

genome

Composed of promoter site and operator site

Controlling site of operon

attachment for RNA Polymerase

promoter site

serves as attachment for repressor protein

operator site

a. Includes structural gene that codes for a repressor protein

Regulatory Operon

b. Includes a promoter site that serves as an attachment for RNA polymerase

Regulatory Operon for Repressor Protein

c. Has no operator site (no repressor protein for the repressor protein ha!)

Regulatory Operon for Repressor Protein

1. The substrate binds to the repressor protein 2. The repressor protein changes shape and has less affinity for operator site 3. Now RNA polymerase can attach to promoter region; transcribe the structural genes into mRNA

Inducible enzymes

1. If the substrate is absent, the repressor protein is in the wrong shape to bind/block the operator site 2. Enzymes are produced 3. Product is produced 4. The produce forms a complex with repressor protein 5. The product-repressor complex fits the operator site and blocks RNA polymerase 6. – no more transcription.

Repressible enzymes

a. DNA supercoiling is relaxed by a. DNA helix unwinds and unzips b. Synthesis of complementary strands

DNA replication steps

i. Topoisomerase in eukarya | ii. DNA gyrase in prokarya

DNA supercoiling relaxed by

i. Starts at a single “origin of replication” in prokaryotes 1. Unzips in both directions ii. (Multiple origins in Eukarya) iii. Both catalyzed by helicase

dna helix unwinds and unzips

i. Each strand of DNA acts as a template for the arrangement of complement nucleotides 1. Remember A to T and C to G

complementary strands

builds RNA primer that’s necessary to start the complementary strand

RNA Polymerase

1. Takes over after the primer is built 2. Catalyzes the addition of DNA nucleotides to the growing DNA molecule a. Archaea have a special DNA polymerase i. Used in PCR

DNA Polymerase

1. The fragments are ultimately attached to one another via an enzyme called a ligase 2. DNA Pol can only add to a strand on its 3’ end, meaning it “writes” from 5’ to 3’ a. Has to then read a strand from 3’ to 5’

DNA replication is continuous on the 3’ strand and discontinuous on the 5’ strand

1. Unzips the DNA helix

Helicase

1. Synthesizes an RNA Primer

Primase/RNA Polymerase

1. Adding bases to the new DNA chain | 2. Proofreads the chain for mistakes

DNA Polymerase III

1. Removes RNA primer 2. Closes gaps 3. Repairs mismatches

DNA Poly I

1. Final binding of nicks in DNA during synthesis and repair

Ligase

1. Supercoiling

Gyrase

i. Source: synthetic agents ii. Effect: block DNA gyrase (the enzyme responsible for relaxing the DNA supercoiling prior to the unwinding of a DNA strand) 1. Prevents relaxation of the DNA

a. Naladixic acid and ciprofloxacin | Interfere with DNA Replication

i. Source: synthetic nucleotide mimic that has a base similar to guanine ii. Target: DNA Polymerase iii. Effect: Competitive inhibition of DNA synthesis iv. Microbe affected: 1. Genital Herpes 2. Chickenpox 3. Shingles v. Toxicity 1. Brain seizures 2. Confusion and skin rash

Interfere with DNA Replication | Acyclovir

i. = Azidothymidine (and related synthetic drugs) ii. Source: 1. Synthetic thymine analogue a. Mimics thymine nucleosides iii. Target: 1. Reverse transcriptase a. (has a lesser affinity for DNA Polymerase) iv. Effect: 1. Competitively inhibits the transcription of HIV RNA into HIV DNA v. Microbe Affected 1. Retroviruses (like HIV – the AIDS virus) vi. Toxicity 1. Causes anemia and immunosuppression a. Inhibits mitochondrial DNA replication

Interfere with DNA Replication | AZT

are antiviral drugs that are analogs of purines and pyrmidines (nucleoside mimics)

AZT and Acyclovir

how to make mRNA

transcription

i. DNA untwists and unzips to expose the codons ii. RNA polymerase attaches to promoter site on one of the DNA strands 1. Specifically the template strand, which is the strand from which mRNA can be generated continuously iii. Complementary RNA nucleotides are attached sequentially until a “stop” codon is reached iv. There are no introns to remove in prokaryotes and finished mRNA may represent more than one protein v. Completed m-RNA

Transcription

fold introns, remove them and splice remaining exons together.

Spliceosomes | Eukaryotes

a. mRNA Sent out of the nucleus into the cytoplasm where it will encounter 80s ribosomes

Eukaryotes | Completed mRNA

can be translating the same m-RNA at the same time

Polyribosomes

i. Happens as the the m-RNA is being

70s ribosomes find and attach to mRNA | Prokaryotes

v. There are no introns to remove in prokaryotes and finished mRNA may represent more than one protein

fact.

2. A transfer RNA (tRNA) with an appropriate complementary anticodon and carrying an activated amino acid attaches at the start codon

Translation

1. Begins when 30s ribosome subunit binds to the start codon (AUG) on the m-RNA

Initiation | Translation

1. the transcript RNA 2. Consists of a single chain (half a ladder) of nucleotides a. Has the same bases as DNA except Thymine replaced by uracil b. Uses the sugar ribose instead of deoxyribose

mRNA

1. derived from a large precursor RNA molecule that is split into not only ribosomal RNA, but also transfer RNA 2. Combines with protein to form the 50s and 30s subunits of ribosomes

rRNA

1. made from a number of different precursor RNA molecules 2. consists of about 80 nucleotides with one exposed codon a. this is called the anti-codon 3. is the “decoder” molecule a. transports ATP activated amino acids to the appropriate, complementary codon on the mRNA. b. Responsible for the translation of the mRNA code into an actualy amino acid sequence

tRNA

1. Can be doing transcription/translation at the same time 2. Do not have introns and exons 3. Finished mRNA may represent more than one protein

Prokaryotes

1. Have removal of introns via spliceosomes | 2. Transcription and translation are different times and location (nucleus vs. cytoplasm)

Eukaryotes

a. E i. Linear chromosomes b. B i. 1 circular c. A i. 1 circular

DNA

a. E i. histones b. B i. none c. A i. Histone like

Histones

i. Introns in m-RNA ii. Operons rare iii. Topoisomerase (relaxes the supercoiling) iv. Replicates through mitosis

i. no introns in m-RNA ii. Operons iii. DNA gyrase relaxes the supercoiling iv. Binary fission for replication

i. No introns in mRNA ii. DNA gyrase for relaxing the supercoiling iii. Has Unique DNA Polymerase that tolerates extreme temperatures

i. M-RNA processing in the nucleus | ii. Transcription is separate from translation

i. One class has only one type of RNA polymerase; the other has >1 ii. No m-RNA processing iii. Transcription and Translation happen at the same time.

B+A

a method for rapidly generating identical strands of DNA

Polymerase Chain Reaction

b. Heating a DNA strand of 94 degrees C for 30 seconds, | i. causes the DNA to unwind and unzip

PCR

c. Cooling the DNA to 50 degrees C | i. will allow primers to attach to the two separate DNA strands

PCR

d. Raising the temperature to 72 degrees C for 2 minutes and providing DNA polymerase i. promotes the synthesis of complementary strands

PCR

i. Provide the unique heat resistant DNA Polymerase used for this

Archaea | PCR

a. Involve environmental mutagens i. Viruses ii. UV radiation 1. Dimers are the most common mutations iii. Compounds foreign to the cell

Induced mutation

a. Involve DNA Replication errors

Spontaneous Mutation

a. When, within a codon, one nucleotide is replaced by another with a different base b. If the new codon codes for the same amino acid as the original codon, no change in he protein coded by the gene will occur c. If the substitution results in a code for a different amino acid, the resultant protein may be defective i. Hemoglobin in sickle cell anemia differs from normal hemoglobin by one aino acid

Point mutation | Base Substitution

a. Due to insertions or deletions of nucleotides b. Can result in changing all the codons that follow the insertion or deletiono c. Change the encoded protein COMPLETELY

Point mutation | Frameshift

1. Found naturally in sunlight 2. Wavelengths of 40nm-390nm 3. Causes the redistribution of electrons and protons in thymines and cytosine, making them highly reactive a. Bonding of adjacent thymines and/or cytosines creates dimers, which distort the DNA b. Interferes with DNA replication and transcription c. Most common mutation***

Non Selective Agents that Damage DNA | UV light

a. Create hyperactive ions which react with nucleotides causing the release of bases and the breakage of the DNA b. Create free radicals i. Highly reactive molecular fragments that have an unpaired electron; attack DNA c. Have high penetration, but sterilization is often not possible because of negative effects on the media d. Kill salmonella and E. coli in food products e. can be used to sterilize plastic equipment and pharmaceutical products, as well as preventing food spoilage

Non Selective Agents that Damage DNA Ionizing radiation Gamma rays

a. Can create free radicals that attack DNA | b. Have considerable energy and penetration

Non Selective Agents that Damage DNA Ionizing Radiation x rays

1. Increase the penetration power of water | 2. Are either anionic (negatively charged) or cationic (positively charged)

Non Selective Agents that Damage DNA | Soaps and detergents

i. Usually bad

mutations

ii. Discovered by Barbara McClintock in 1948.

Other sources of genetic variation | transposons

i. Special DNA segment that have the capability of moving from one location in the genome to another – “jumping genes.” ii. Discovered by Barbara McClintock in 1948. iii. Causes the rearrangement of the genetic material iv. Can move from one chromosome site to another, from a chromosome to a plasmid, or from a plasmid to a chromosome. v. May be beneficial or harmful vi. Thought atht 45% of our genome could be from these. vii. A “jumping gene” that can “jump” between chromosomes or plasmids viii. Enters the host genome via the enzyme transposase generating recombinant DNA (DNA generated from two or more sources)

Other sources of genetic variation | transposons

a. Where plasmids are transferred via a mating bridge involving sex pili in G- cells i. attaches to a receptor on a target cell retracts, pulling the cells together

Other sources of genetic variation | conjugation

1. Plasmids are… a. G+ cells gain intimate contact and exchange DNA through a conjugation bridge b. donor transfers a copy of plasmid through pilus

Other sources of genetic variation | conjugation

a. A copy of the chromosome can begin the passage to a second cell via mating/conjugation bridges b. The passage is usually interrupted and the chromosomes breaks such that only a partial transfer occurs c. Transferred chromosomal DNA can integrate itself into the new cell’s genome, creating recombinant DNA

Other sources of genetic variation conjugation involving chromosomes

3. Conjugation can be accelerated when the cells are exposed to environmental stress

Other sources of genetic variation conjugation truth

1. = the transfer of genes from one bacterium to another via a virus=bacteriophage

Other sources of genetic variation | transduction

a. Some viruses can infect several species and even genera

Other sources of genetic variation

a. Random fragments of disintegrating host DNA are picked up by the phage during assembly i. Any gene can be transmitted this way b. The bacterial genome breaks into fragments and some of the newly forming viruses pick up a bacterial DNA fragment instead of viral DNA c. Bacterial DNA is then injected into another bacteria

Other sources of genetic variation | Generalized Transduction

a. A highly specific part of the host genome is regularly incorporated into this virus b. Viral DNA is integrated into a bacterial genome at one specific site i. =lysogenized c. The virus excises itself along with the neighboring bacterial DNA i. A restricted group of bacterial genes are transferred to new host cell, instead of a random fragment.

Other sources of genetic variation | Specialized transduction

b. Viral DNA is integrated into a bacterial genome at one specific site

Other sources of genetic variation transduction =lysogenized

1. Chromosome fragments from a lysed cell are accepted by a recipient cell; the genetic code of the DNA fragment is acquired by the recipient a. Donor and recipient cells can be unrelated b. Useful tool in recombinant DNA technology

Other sources of genetic variation | Transformation

2. Where Naked DNA is taken up by bacteria and recombines with the main bacterial genome

Other sources of genetic variation | Transformation

the term used when eukarya take up naked DNA

Other sources of genetic variation | Transfection

a. For bacteria, competency can occur for a short time during the log phase of growth b. Is facilitated by a competence factor that can be released into the medium by the bacterial cells or displayed as a surface protein that binds to the DNA

Cells must be competent in order to take up naked DNA

i. Live strain w/ capsule killed rat ii. Heat killed strain/w capsule rat survives iii. Live strain, no capsule rat lives iv. Live no capsule + dead capsule = dead rat 1. Both strains are found in dead rat LIVING with isolated

Griffith's work Transformation Streptococcous Pneumoniae

1. When genes from a cell are transferred to another existing cell a. i.e. when genes from a bacterium are transferred to a neighboring bacterium

Other sources of genetic variation | Lateral Gene Transfer

is when genes are passed to two daughter cells that result from mitosis or binary fission

Other sources of genetic variation | Vertical Gene Transfer

2. Conjugation, transduction, and transformation (transfection)

Other sources of genetic variation | all result in lateral gene transfer

3. About 80% of microbe genes have been laterally transferred at some point in their history. 4. Bacterial speciation is more driven by LGT than from mutations. Pretty cool!

i. =the intentional removal of genetic material from one organism and combining it with that of a different organism ii. Bacteria picks up plasmid by trransformation

Recombinant DNA Technology

1. Objective is CLONING, which requires that the desired donor gene be selected, excised by restriction endonucleases, and isolated 2. The gene is inserted into a vector (plasmid, virus) that will insert the DNA into a cloning host 3. Cloning host is usually bacterium or yeast that can replicate the gene and translate it into a protein product.

Recombinant DNA Technology

A gene source i. A vector = what you use to transfer the gene to the bacteria ii. A cell in which the recombinant can proliferate =clone

Genetic Engineering | requirements

1. The gene must include a start codon, a stop codon, AND a controlling site 2. Gene product must not be toxic to a host cell

Genetic Engineering | gene source

1. DNA into which the desired gene is spliced so the gene can be inserted into a host cell a. Since host cells won’t usually accept a foreign gene all by itself

Genetic Engineering | vector

2. bacterial and yeast plasmids as well as viral DNA are often used a. must be able to replicate b. must have a gene that allows selection of the DNA that is recombinant i. usually two genes for antibiotic resistance

Genetic Engineering | vector

= viruses, have the natural ability to inject their DNA into bacterial hosts through transduction

Genetic Engineering | = bacteriophages

iii. A cell in which the recombinant can proliferate

Genetic Engineering | =clone

i. Construction of the recombinant DNA ii. Introduction of the recombinant into the host cell via a vector iii. Selection of a host cell with a recombinant DNA iv. Cloning and expression of the gene involves

Steps in Genetic Engineering

a. Bind DNA (DNA in the vector as well as well as DNA in the gene source) and uct it in one or more places at specific sites i. Each species or strain of bacteria has a specific restriction endonuclease that normally breaks down foreign DNA

Steps in Genetic Engineering i. Construction of the recombinant DNA Restriction endonucleases

a. Cuts DNA so it has staggered ends (sticky ends) | b. is used to prepare DNA so a gene can be spliced in to generate recombinant DNA

Steps in Genetic Engineering i. Construction of the recombinant DNA Restriction endonuclease

is used as a vector to carry and incorporate genes into mammalian cells

Steps in Genetic Engineering ii. Introduction of the recombinant into the host cell via a vector viral genome

a. are the most common techniques for delivering “naked DNA” to new hosts b. The host cells need to be made competent c. Eukaryotic plant cells can be transfected (infected by the new recombinant DNA) by the gram negative bacteria Agrobacterium tumefaciens

Steps in Genetic Engineering ii. Introduction of the recombinant into the host cell via a vector Transformation of bacterial cells Transfection of eukaryotic cells

1. Are usually made competent via exposure to chloride salts solutions and temperature extremes or via electroporation a. Cells are exposed to pulses of electrical fields that open small pores in cell membranes 2. Plasmids are used as vectors to transform bacteria

Steps in Genetic Engineering ii. Introduction of the recombinant into the host cell via a vector Truth.

1. The vector, with the gene of interest, needs to include two genes for antibiotic resistance

Steps in Genetic Engineering | iii. Selection of a host cell with a recombinant DNA

2. The host cells are plated on a media that contains the 2 antibiotics

Steps in Genetic Engineering | iii. Selection of a host cell with a recombinant DNA

3. Cells with appropriate antibiotic resistance, that is, resistance to 1 of the antibiotics (and therefore has the gene of interest) can be identified

Steps in Genetic Engineering | iii. Selection of a host cell with a recombinant DNA

1. Harvesting the resistant cells that contain the recombinant (gene of interest) 2. Allowing the cells to multiply (gene amplification)

Steps in Genetic Engineering | iv. Cloning and expression of the gene involves

Significance of Genetic Engineering 1. Factor VIII a. For hemophilia 2. Growth hormone and insulin 3. The gene for normal hemoglobin 4. The gene for normal chloride ion channels a. Cystic fibrosis 5. Interleukin II (Bubble Boy) for the treatment of SCID (Severe Combined Immunodeficiency)

i. Genetically engineer human proteins and cells to correct hereditary diseases

Significance of Genetic Engineering 1. Alpha interferon for treatment of Hairy Cell Leukemia 2. Vaccines a. Hepatitis B (HBV) i. The gene for a viral surface protein is inserted into a yeast plasmid

Genetically engineered drugs

Significance of Genetic Engineering | = use of an organism’s biochemical and metabolic pathways for industrial production

Biotechnology

Significance of Genetic Engineering i. Gene for herbicide resistance ii. Bacteria transformed iii. Transfection of plant cells iv. 99% of corn, soy, and cotton are genetically engineered.

eukaryotic plants can be transfected by the G- bacteria, Agrobacterium Tumefaciens, plant pathogens

Significance of Genetic Engineering | i. Correct or repair a faulty gene in humans

gene therapy

Significance of Genetic Engineering | a. Normal gene cloned in vectors, tissue removed from the patient

ex vivo

Significance of Genetic Engineering | a. Naked DNA or vector is directly introduced to the patients tissue

in vivo gene therapy

1. Glycolysis ii. Incomplete oxidation of glucose/carbs in the absence of oxygen iii. Yields small amount of ATP 1. But less competition in areas w/o oxygen iv. Formation of acid, gas, and other products by action f various bacteria on pyruvic acid v. Glycolysis 1. Organic compounds are the final electron acceptors vi. Products include 1. Swiss cheese with CO2 a. Makes the holes

Fermentation

i. Glycolysis ii. Kreb’s cycle iii. Respiratory chain iv. Yields about 28ATP

Aerobic Respiration

i. Glycolysis ii. Kreb’s cycle iii. Respiratory chain 1. Molecular Oxygen is NOT the final electron acceptor 2. Has oxygen containing ions but not free oxygen 3. Nitrate (NO3) and nitrite (NO2-) iv. Yields fewer ATP (~20)

Anaerobic Respiration

1. O2- 2. Superoxide 3. Free radical

i. All organisms exposed to oxygen produce the toxic molecule

ii. Obligate aerobes plus facultative and microaerobes have enzyme which converts O2- into

hydrogen peroxide

a. Breaks O2- into hydrogen peroxide

Superoxide dismutase

a. Breaks hydrogen peroxide into H2O and O2

Catalase

i. Anaerobic and can be independent of the sun ii. Can occur in extreme temperatures and pressures iii. H2 oxidised to form sugar iv. Sulfur compounds are oxidized to form sugar v. Rocks are oxidized to form sugar

chemosynthesis

1. Methane as byproduct 2. Found in variety of ecological niches a. Seqage slude b. Marine and lake sediments c. Geothermal springs d. Deep sea hydrothermal vents e. Animal intestines (cow farts) f. Archaea photosynthesis

Methanogens

i. Archaea 1. Bacteriorhodopsin ii. Only produces ATP iii. No Calvin Benson Cycle – no sugar iv. No ETC

Archaea photosynthesis

Light dependent reactions

Oxygenic photosynthesis

1. Light a. Cyclic Phosphorylation i. ATP 1. +CO2 2. Calvin Benson Cycle (light indp) 3. Sugar b. +H2O + NADP i. Non Cyclic Phosphorylation 1. ATP/NADPH/O2 a. Calvin Benson Cycle Sugar/H2O/RuBP

Oxygenic photosynthesis | ight dependent reactions

i. Done by green and purple sulfur bacteria ii. Uses the pigment bacteriochlorphyll iii. Uses hydrogen sulfide or hydrogen gas for replacement electrons iv. No O2 released

Anoxygenic photosynthesis

i. Breakdown of Glucose to pyruvic acid ii. For one glucose, 1. 2 ATP IN, 4 ATP out = net 2ATP gain 2. 2 NADH (for electron transport chain) 3. 2 pyruvic acid

Glycolysis

i. Pyruvic acid  Acetyl CoA before begins this ii. Processes pyruvic acid and generates iii. 3 CO2 molecules iv. NADH and FADH2 generated v. ATP vi. Makes 6 NADH, 2FADH2 (per glucose; goes to ETC) vii. Makes 2 ATP and 6CO2 (per glucose)

Kreb's cycle

i. Final processing of electrons and hydrogen and the major generator of ATP ii. Chain of redox carriers that receives electrons from reduced carriers (NADH and FADH2) iii. Shuttles electrons down the chain 1. Energy released 2. Subsequently captured iv. Used by ATP synthase complexes to produce ATP 1. Oxidative phosphorylation v. Accepts electrons from NADH and FADH vi. Generates energy through sequential redox reactions called “oxidative phosphorylation” vii. It’s a total of 10 NADH and 2FADH2/glucose give electrons and hydrogens to the ETS 1. Electrons pass along a group of coenzymes 2. Hydrogens pumped to to exterior of the cell membrane, creating a gradient viii. NADH  3 ATP ix. 1 FADH2  2 ATP

Electron Transport System | + Phosphorylation

1. Algae and Plants | a. Chlorophyll a and b

Oxygenic photosynthesis pigment

1. Cyanobacteria a. Phycobiliprotein b. Chlorophyll a and b Sometimes

photosynthesis pigment bacteria oxygenic photosynthesis

a. Bacteriochlorophyll

photosynthesis pigment bacteria anoxygenic photosynthesis

i. Archaea 1. Bacteriorhodopsin ii. Only produces ATP iii. No Calvin Benson Cycle – no sugar iv. No ETC

photosynthesis pigment | Archaea photsynthesis

i. The ultimate source of all the chemical energy in cells comes from the sun 1. 6CO2 + 6 H2O –light glucose (C6H12O6)+6O2

Photosynthesis in general

1. Electron released from pigment returns to its original photosystem (photosystem 1) via the ETS 2. Only generates ATP

Cyclic photophosphorylation

1. If electrons lost from PS1 are picked up by a molecule of NADP 2. The electrons are released from chlorophyll in PSII a. Used to replace those lost by chlorophyll in PS 1 b. Are delivered to PS1 via ETS that generates ATP 3. H2O provides final replacement electrons (and O2) a. Photolysis of H2O with loss of flectrons to PSII b. PSII powerful oxidizing agent, removes electrons from hydrogens in water i. Electrons are used to replace electrons lost form chlorophyll in PS II ii. ATPs generated in ETC iii. H+ are used for the reduction of NADP- to NADPH 4. Generates ATP and NADPH to go to the Calvin Benson cycle

noncyclic photophosphorylation

i. Light independent | 1. Uses ATP and NAPH to fix CO2 and convert it to glucose

Calvin Benson cycle

1. Must obtain carbon in an organic form made by other living organisms a. Proteins b. Carbohydrates c. Lipids d. Nucleic acids

Carbon Source | Heterotroph

1. =self feeder 2. An organism that uses Co2, an inorganic gas, as it’s carbon source 3. Not nutritionally dependent on other things 4. Makes its own sugars

Carbon source | Autotroph

1. Gains its energy from chemical compounds | a. Glycolysis and breaking down glucose

Energy source | chemotroph

1. Gain energy through photosynthesis

Energy source | phototroph

1. Don’t use light so have to each other things as a source of nutrients 2. Have organic compounds as its carbon source 3. Differ based on their final electron acceptor

Chemoheterotrophs

Chemoheterotrophs i. All animals ii. Most fungi iii. Protozoa iv. Bacteria

Chemoheterotrophs | O2 electron acceptor

Chemoheterotrophs i. Separated by ii. Inorganic compounds 1. Like Clostridium iii. Organic compounds 1. Fermentation of Streptococcus for instance

Chemoheterotrophs | No O2 electron source

1. The ones that do chemosynthesis 2. Energy source chemicals 3. Carbon source CO2 4. Archaea and Bacteria

Chemoautotrophs

1. Light as energy source 2. Organic compounds for C source a. Green and purple, nonsulfur bacteria b. Archaea

Photoheterotrophs

1. Light energy source 2. Co2 carbon sourc 3. Use H2O to reduce CO2?

Photoautotrophs

Photoautotrophs i. Oxygenic photosynthesis 1. Plants/algae 2. cyanobacteria

Photoautotrophs | Use H2O to reduce CO2

Photoautotrophs i. Anoxygenic photosynthesis 1. Green purple sulfur bacteria

Photoautotrophs | Do not use H2O to reduce CO2

1. Lowest temperature that permits a microbe’s growth and metabolism

Minimum temperature

1. Highest termperature that permits a microbe’s growth and metabolism

Maximum temperature

2. Promotes the fastest rate of growth and metabolism

Optimum temperature

i. Optimum temperature 1. 15 degrees C 2. Capable of growth of 0 degrees C Refrigerator spoilage

psychrophile

i. Optimum temperature 20-40degrees | ii. Most human pathogens

mesophile

i. Optimum temperature greater than 45 degrees | ii. compost

Thermophile

i. A mesophile that can grow at 0 degrees | ii. Cause for disease and spoilage

Psychrotolerant

i. Greater than 80 degrees | ii. Includes Extremophiles (which are happy <0 degrees C)

Hyperthermophiles

80 degrees C

Extremophiles

i. Cannot grow without oxygen | ii. Can utilize oxygen and detoxify it

Obligate aerobe

i. Utilizes oxygen but can also grow in its absence 1. Staphylococcus 2. E. Coli

Facultative Anaerobe

i. Requires ony a small amount of oxygen | ii. Helicobacter species

Microaerobes

i. Lacks the enzymes to detoxify oxygen so cannot survive in an oxygen environment

Obligate Anaerobe

i. All organisms exposed to oxygen produce the toxic molecule 1. O2- 2. Superoxide 3. Free radical

truth

i. Oxygen level at which organism switches from aerobic to anaerobic metabolism

Pasteur Point

ell environment results in cellular dehydration and plasmolysis with protein precipitation

High solute concentration

a cell’s environment result in water entering the cell such that cells may lyse

Low solute concentration

1. Require an environment with high concentration of organic molecule 2. Sugar

osmophiles

1. Do not require high concentration of solute but can tolerate it when it occurs

osmotolerant

1. Require environment with high salt concentrations to stabilize their membranes

halophiles

1. Parent cell enlarges, duplicates its chromosomes, and forms a central transverse septum dividing the cell into two daughter cells.

binary fission

a. = Time required to complete fission cycle (one parent  two daughter cells

generation time for bacteria

i. “flat” period of adjustment ii. Enlargement iii. Little growth 1. Cells are increasing in cell mass

Lag phase

i. Period of maximum growth ii. Continues as long as cells have adequate nutrients and a favorable environment iii. Proportional to the rate of energy metabolism iv. Very susceptible to antibiotics and other chemicals at this point.

Log phase | Exponential Growth phase

i. Rate of cell growth = rate of cell death ii. Caused by depleted nutrients and O2, iii. Caused by excretion of organic acids and pollutants iv. Cells in survival mode, making defensive proteins

Stationary Phase

i. As limiting factors intensify, cells die exponentially | ii. Remain are those most resistant and endospores

Death Phase

i. Most simple ii. Turbidity = degree of cloudiness 1. Reflects the relative population size

Turbidometry

i. Dilutions and plating allow plate to have accurate counting of plates ii. Remember the dilutions and you can multiply that and see how many colonies were in the original sample

Plate count

1. Reduce the number of pathogens off inanimate objects | reduces number of microbes

Disinfectant

1. To reduce or inhibit microbes on living tissue | reduces number of microbes

Antiseptic

1. Removes or kills all microbes | a. They are incapable of reproducing

Eliminates microbes

You sterilize PLUS remove all microbial toxins

Decontaminations

i. Inhibits bacteria

Bacteriostatic

Kills bacteria

Bacteriocide

Kills fungi

Fungicide

Kills microbes

Germicide

Destroys bacterial or fungal spores

Sporicide

i. Sterilizes | ii. Like what we do to inoculating loop and inoculating needle

Dry heat | oven and incineration

1. Sterilizes if spores are not present

Boiling

1. Sterilizes if spores are present

Tyndallization

1. Pressure cooker 2. Results in temperatures about boiling 3. Good penetration 4. The most practical and dependable 5. Sterilizes, DOES NOT CONTAMINATE

Autoclave

1. Does not sterilize 2. Used to eliminate pathogens from food products a. Usually beverages like milk 3. Controlled heat BELOW boiling (72 degrees for milk) for 15 seconds 4. Media that are concentrated or contain fats and sweeteners require higher temperature a. Skim milk vs. cream

Pasteurization

i. Shortest time required to kill all microbes in a suspension at a given temperature.

Thermal Death Time

i. The lowest temperature at which microbes are killed in 10 minutes

Thermal Death Point

i. Bacteria are added to tubes with different dilutions of a chemical agent and then incubated ii. Identify agents that prevent growth at the great dilution iii. Minimal inhibitory concentrations 1. Serial dilution of the agent plus a. A suspension of the organism 2. = the tube with the lowest amount of agent that is without visible growth

Dilution tests

i. Filter paper method ii. Look for a clear area around the paper disks soaked in a given agent. 1. This is where bacteria growth has been inhibited iii. The media used in determining sensitivity should be comparable to tissue fluids of the body iv. Staphylococcus Aureus is the usual test organism v. Can’t tell if the organisms are dead or inhibited vi. Various agents diffuse through agar at differet rates

Sensitivity Discs

i. Occasionally acquired during spontaneous mutation in critical chromosomal genes ii. OR iii. Acquisition of new genes or sets of genes via transfer from another species. 1. Originates from resistance factors (plasmids) encoded with drug resistance, transposons iv. Survival when there was a high concentration of antibiotics

How resistance of genes are spread

a. ajor contributors to resistance i. Hospitals (immunosuppressed, sick, old) ii. Daycare centers (immature immune systems, high exposure) iii. Antibiotic use in livestock iv. Third world countries (no controls on use, war, and poverty) v. First world countries 1. Given unnecessarily 2. Instructions not followed 3. Antibiotics in products

Where do humans most often encounter resistant microbes?

Immunocompromised Kids Health care workers

who's at risK?

a. Produce enzymes that activate antibiotics b. Prevent antibiotics from reaching the target c. Alter antibiotic targets d. Change the enzyme’s pathway e. Note that resistance tends to increase with time f. Humans pool antibiotics from trillions of bacteria

Major resistance strategies by microbes

ii. Actual results are the same when treating livestock with antibiotics such that we can acquire resistant bacteria from animal products. Body flora of livestock represent the 3rd major reservoir of genes for antibiotic resistance. iii. The more effective a treatment is at eradicating a susceptible population fof bacteria, the more it will promote the development and spread of resistant bacterial populations.

Successful Antibiotic treatment

i. The sensitive bacterial pathogen and benign normal body flora are both killed by the antibiotic 1. The “shield” that normal body flora provide against new pathogens entering the body is weakened 2. Resistant normal body flora a. Will flourish without competition and create super infections i. E.g. Clostridium difficile b. Surviving resistant normal body flora act as a reservoir for resistance genes that can be transferred to antibiotic sensitive pathogens

Successful antibiotic trreatment

i. Surviving resistant pathogens may replicate leading to a renewal of the disease ii. The antibiotic fails 1. The disease is now harder to treat 2. Re-emergence of disease and an increase in untreatable infections iii. Wrong antibiotic given or wrong dosage iv. Instructions not followed

Unsuccessful antibiotic treatment

i. Hospitals (immunosuppressed, sick, old) ii. Daycare centers (immature immune systems, high exposure) iii. Antibiotic use in livestock iv. Third world countries (no controls on use, war, and poverty) v. First world countries 1. Given unnecessarily 2. Instructions not followed 3. Antibiotics in products

Major contributions to resistance

i. Increased health costs ii. Re-emergence of disease iii. Loss of work iv. Higher death rates v. Increased side effects

Consequences of antibiotic resistance

a. Use less antibiotics! b. Practice prevention c. Reduce exposure in hospitals and daycares d. Survey diseases and resistance e. Eliminate vectors that spread disease f. Educate people (and doctors) on the proper use of antibiotics

Best strategy to reduce number of resistance bacterial populations

a. Because resistance is on a plasmid, it slows replication rate. b. Would be a disadvantage in an environment without anti biotics c. Allows the sensitive bacteria to outgrow and outcompete the resistant ones d. Loss of plasmids becomes favored (plasmids not copied, unevenly distributed to daughter cells). e. Resistance still maintained but on a lower level.

Explain why resistant bacteria are disadvantaged when antibiotic use is discontinued.

Exam 2 Super Review Flashcards

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